Chronic myeloid leukemia (CML) is a form of cancer characterized by the increased proliferation and accumulation of myeloid cells in the bone marrow and blood. CML represents 14% of all leukemia cases and 33,400 new cases of CML were reported in 2004. CML is associated with chromosomal translocation between chromosome 9 and 22 resulting in formation of characteristic Philadelphia chromosome. Pathologically significant end product of chromosomal translocation is fusion of two genes, BCR (breakpoint cluster region) gene from chromosome 22 and ABL gene on chromosome 9. This abnormal fusion results in formation of chimera and constitutively active protein known as BCR-ABL, a tyrosine kinase receptor which is responsible for uncontrolled cell cycle and other cancer related biological features. BCR-ABL thus plays a role in induction of cancer during CML and hence acts as a therapeutic target of CML (Bartram, et al., Nature, 306:277 (1983)).
A series of inhibitors, based on the 2-phenylaminopyrimidine class of pharmacophores, has been identified that have exceptionally high affinity and specificity for ABL (Zimmerman, et al., Med. Chem. Lett., 7:187(1997)). Imatinib mesylate (also known as STI-571 and Gleevec) is an orally bioavailable 2-phenylaminopyrimidine with targeted inhibitor activity against the constitutively active tyrosine kinase of the BCR-ABL chimeric fusion protein (Quintás-Cardama, et al., Mayo Clin. Proc., 81:973 (2006)). Nilotinib (also known as AMN107) is a phenylamino-pyrimidine derivative with a 20- to 30-fold increased potency compared with imatinib against BCR-ABL. This may be due to a better fit within the ABL kinase pocket. However, nilotinib, like imatinib, only binds the ABL protein in its inactive conformation. Imatinib has become the first drug of choice CML treatment. Despite the success of imatinib and nilotinib, a significant number of patients (10-30%) have demonstrated poor initial response (Drucker, et al., Nature Med. 2:561 (1996)). During the course of the disease, some percentage of CML patients develops mutations in the ATP binding domain of the BCR-ABL tyrosine kinase which make them resistant to imatinib therapy. Because of the tighter binding to BCR-ABL, nilotinib can be used for treatment of patients who respond weakly to imatinib or imatinib resistant patient. However in some cases, patients may develop resistance to both nilotinib and imatinib. Discovery of resistance to imatinib and nilotinib therapy has led to the emergence of new drugs and therapeutic strategies. Therefore, there is significant need to develop means of rapidly monitoring the effects of imatinib or nilotinib at early stages.
Identification of BCR-ABL mutants that are resistant to imatinib and/or nilotinib has become an integral part of patient care management in CML therapy yet current diagnostic and prognostic tools are too expensive, time consuming, and require highly skilled labor. The approach described herein is a rapid, easy-to-use immunochromatography-based testing method utilizing drug immobilization to detect drug-protein interactions.